4.1 Efficient design of in vivo cascades
We developed a production strain harboring an efficient cascade for the
conversion of cyclohexane to 6HA with a decent activity in the
50-60 U gCDW-1 range. It has been
shown that detailed analyses of enzyme kinetics and respective reaction
engineering for a three-step cascade could efficiently enhance the
conversion of several unsaturated cyclic alcohols to the corresponding
lactones in vitro [1, 30]. Scherkuset al. analyzed the kinetic parameters of an alcohol
dehydrogenase and a CHMO to produce 6HA from cyclohexanol[31]. Similarly to the CDH investigated in our
study, the KM value was significantly higher for the
reverse reaction, and CHMO was severely inhibited by cyclohexanol.
Establishing a kinetic model enabled the setup of an efficient fed-batch
process.
Whereas the balancing of enzyme ratios in vitro is a rather
straight-forward approach [1], in case of
whole-cell biocatalysis, this requires fine-tuning of expression levels
which, furthermore, should not drive the demand of resources beyond
cellular capacities [7]. The so-called metabolic
burden arises from the change in demand for (biomass) building blocks
and energy (ATP, NAD(P)H) and is system- and condition-dependent[32, 33]. In this study, we observed a gradual
decrease in the growth rate with increasing operon size (Table S4). For
the cascade investigated, the two-operon- compared to the one-operon
approach not only enabled faster growth indicating low metabolic burden,
but also led to higher CDH and CHMO expression levels and cascade
activities. The relation between gene organization and gene expression
is poorly understood. It has been found for E. coli that gene
expression increases with the length of the operon resulting in more
cotranscriptional translation [34]. Increased
translation can result in metabolic burden and misfolded or otherwise
non-functional proteins, which was found for the Cyp in our previous
study [18]. Although without a terminator after
the Cyp genes (Figure 3A), RNA polymerase dissociation may have been
promoted by the transcription initiation machinery occupying the
downstream promoter region and thereby opening up the DNA[33]. Thus, mRNAs with shorter average length can
be expected for the two promoters- as compared to the one promoter
constructs. Shorter mRNAs, in turn, have been found to show increased
stability in E.coli cells [35] and to
recruit fewer ribosomes [34], thus decreasing the
metabolic burden. In general, the metabolic burden increases with gene
and operon size and with the plasmid copy number. It is further enhanced
by some antibiotics such as kanamycin and thus tends to be high for
plasmid-based expression, especially when antibiotic resistance genes
are used as selection markers [36]. The two operon
approach may have profited from shorter but more stable mRNAs and thus
reducing metabolic burden and can be considered suitable for efficient
expression of the designed pathways in P. taiwanensis VLB120. For
further optimization, metabolic modeling of cascades and combinatorial
pathway engineering taking into account metabolic burden effects may
become interesting, although they still suffer from incomplete knowledge[37-39].